Device and method for high-throughput quantification of mRNA from whole blood

ABSTRACT

Disclosed are a method, device kit, and automated system for simple, reproducible, and high-throughput quantification of mRNA from whole blood. More particularly, the method, device, kit and automated system involve combinations of leukocyte filters attached to oligo(dT)-immobilized multi-well plates.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/796,298, filed on Mar. 9, 2004, now U.S. Pat. No. 7,745,180 B2, whichis a continuation-in-part of U.S. application Ser. No. 10/698,967, filedon Oct. 30, 2003, now abandoned, which is a continuation-in-part ofInternational Application No. PCTUS03/12895, which was filed in Englishon Apr. 24, 2003, and was published in English, and claims the benefitof U.S. Provisional Application No. 60/375,472, filed on Apr. 24, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to high-throughput isolation andquantification of mRNA from whole blood. More particularly, thisinvention relates to a method and device for isolating and amplifyingmRNA using combinations of leukocyte filters attached tooligo(dT)-immobilized multi-well plates.

2. Description of the Related Art

Research in the field of molecular biology has revealed that the geneticorigin and functional activity of a cell can be deduced from the studyof its ribonucleic acid (RNA). This information may be of use inclinical practice, to diagnose infections, to detect the presence ofcells expressing oncogenes, to detect familial disorders, to monitor thestate of host defense mechanisms and to determine the HLA type or othermarker of identity. RNA exists in three functionally different forms:ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA).Whereas stable rRNA and tRNA are involved in catalytic processes intranslation, mRNA molecules carry genetic information. Only about 1-5%of the total RNA consists of mRNA, about 15% of tRNA and about 80% ofrRNA.

mRNA is an important diagnostic tool, particularly when it is used toquantitatively observe up- or down-regulation of genes. Human peripheralblood is an excellent clinical resource for mRNA analysis. The detectionof specific chimeric mRNA in blood, for example, indicates the presenceof abnormal cells and is used in molecular diagnostics for chronicmyelogenous leukemia (CML) (Kawasaki E. S., Clark S. S., Coyne M. Y.,Smith S. D., Champlin R., Witte O. N., and McCormick F. P. 1988.Diagnosis of chronic myeloid and acute lymphocytic leukemias bydetection of leukemia-specific mRNA sequences amplified in vitro. Proc.Natl. Acad. Sci. USA 85:5698-5702, Pachmann K., Zhao S., Schenk T.,Kantaijian H., El-Naggar A. K., Siciliano M. J., Guo J. Q., ArlinghausR. B., and Andreeff M. 2001. Expression of bcr-able mRNA individualchronic myelogenous leukaemia cells as determined by in situamplification. Br. J. Haematol. 112:749-59). Micrometastatic cancercells can also be detected in blood by measuring cancer-specific mRNA,such as carcinoembryonic antigen (CEA) for colon cancer, prostatespecific antigen (PSA) for prostate cancer, thyroglobulin for thyroidcancer (Wingo S. T., Ringel M. D., Anderson J. S., Patel A. D., Lukes Y.D., Djuh Y. Y., Solomon B., Nicholson D., Balducci-Silano P. L., LevineM. A., Francis G. L., and Tuttle R. M. 1999. Quantitative reversetranscription-PCR measurement of thyroglobulin mRNA in peripheral bloodof healthy subjects. Clin. Chem. 45:785-89), and tyrosinase for melanoma(Pelkey T. J., Frierson H. F. Jr., and Bruns D. E. 1996. Molecular andimmunological detection of circulating tumor cells and micrometastasisfrom solid tumors. Clin. Chem. 42:1369-81). Moreover, as the levels ofthese cancer-specific mRNA can change following treatment,quantification of specific mRNA provides for a useful indicator duringtreatment follow-up.

As blood contains large quantities of non-nucleated erythrocytes(approximately 5 million cells/μL) compared to leukocytes (approximately5000 leukocytes/μL), the isolation of granulocytes or lymphocytes fromwhole blood is commonly performed as the first step in mRNA analysis.However, due to inconsistencies in the recovery of specific subsets ofleukocytes among different samples, the number of isolated leukocytes isdetermined for each sample and results are expressed as the quantity ofmRNA per leukocytes, not mRNA/μL blood. Moreover, mRNA quantities maychange during lengthy isolation processes. While no method exists forthe isolation of cancer cells from blood, gene amplificationtechnologies enable the identification and quantification of specificmRNA levels even from a pool of different genes, making whole blood anideal material for mRNA analysis when gene-specific primers and probesare available.

The scientific community is facing a huge problem ofinstitute-to-institute and experiment-to-experiment variation in geneexpression analysis, because of the lack of standardization. Althoughrecent gene amplification technologies provide an absolute quantity oftemplate DNA, these values cannot be converted to the amounts of thegene in the original materials, due to the lack of information of theyield of RNA recovery and the efficiency of cDNA synthesis in eachsample. Total RNA is frequently used as a standardization marker formRNA quantitation, and results are typically expressed as the amounts ofgenes per μg total RNA. However, it must be emphasized that total RNAdoes not represent mRNA, because the fraction of mRNA is only 1-5% oftotal RNA, and mRNA volume varies even when the amounts of total RNA isidentical. The yield of total RNA or mRNA also varies widely dependingon which method is employed. Once RNA is extracted, the next step is thesynthesis of cDNA, which itself can create uncertainty since existingmethods do not indicate whether each RNA template creates a single copyof cDNA in each experiment. In order to avoid the above problems,relative quantitation is used widely by comparing the data of targetgenes to that of housekeeping genes or rRNA. However, the amounts ofcontrol genes are typically not consistent and may change duringexperiments. Moreover, this variation presents a serious problem forclinical diagnostics, since each clinical specimen is typically analyzedat a different point in time.

It is typically very difficult to isolate pure mRNA from whole bloodbecause whole blood contains large amounts of RNAases (fromgranulocytes) and non-nucleated erythrocytes. Although various RNAextraction methods are available for whole blood applications (de VriesT. J., Fourkour A., Punt C. J., Ruiter D. J., and van Muijen G. N. 2000.Analysis of melanoma cells in peripheral blood by reversetranscription-polymerase chain reaction for tyrosinase and MART-1 aftermononuclear cell collection with cell preparation tubes: a comparisonwith the whole blood guanidinium isothiocyanate RNA isolation method.Melanoma Research 10:119-26, Johansson M., Pisa E. K., Tormanen V.,Arstrand K., and Kagedal Bl. 2000. Quantitative analysis of tyrosinasetranscripts in blood. Clin. Chem. 46:921-27, Wingo S. T., Ringel M. D.,Anderson J. S., Patel A. D., Lukes Y. D., Djuh Y. Y., Solomon B.,Nicholson D., Balducci-Silano P. L., Levine M. A., Francis G. L., andTuttle R. M. 1999. Quantitative reverse transcription-PCR measurement ofthyroglobulin mRNA in peripheral blood of healthy subjects. Clin. Chem.45:785-89), the assay procedures are labor-intensive, require severalrounds of centrifugation, and involve careful handling that is essentialin eliminating ribonuclease activities.

Consequently, there exists a need for a quick and easy method and devicefor isolating and quantifying large quantities of mRNA from whole blood.Specifically, there exists a need for a high throughput, wholeblood-derived mRNA-processing technology with reproducible recovery anda seamless process to gene amplification.

SUMMARY OF THE INVENTION

The present invention discloses an efficient high throughput method anddevice for isolating and quantifying mRNA directly from whole blood,with reproducible recovery, using combinations of leukocyte filtersattached to oligo(dT)-immobilized multi-well plates.

One aspect of the invention includes a method of high throughputquantification of mRNA in whole blood, including the steps of: (a)collecting whole blood; (b) removing erythrocytes and blood componentsfrom the whole blood by filtration to yield leukocytes on a filtermembrane; (c) subjecting the leukocytes to cell lysis to produce alysate containing mRNA; (d) transferring the lysate to anoligo(dT)-immobilized plate to capture the mRNA; and (e) quantifying themRNA.

In one preferred embodiment of the method, an anticoagulant isadministered to the whole blood prior to collection of leukocytes.Several filter membranes can be layered together to increase the yieldof captured leukocytes. The leukocytes that are trapped on the filtermembrane are lysed using a lysis buffer to release mRNA from theleukocytes. The transfer of lysate to the oligo(dT)-immobilized platecan be accomplished using centrifugation, vacuum aspiration, positivepressure, or washing with ethanol followed by vacuum aspiration. ThemRNA is quantified by producing cDNA and amplifying the cDNA by PCR.Particularly preferred embodiments use TaqMan PCR to quantify mRNA.

Another aspect of the invention comprises the use of artificial controlRNA as a universal standard. In preferred embodiments, measuring therecovery of the standard RNA in each sample allows the results of geneamplification to used to determine the amounts of mRNA present per μl ofwhole blood. Embodiments of the present result in low coefficients ofvariation between samples and experiments. In preferred embodiments,variation can be minimized during RNA recovery, cDNA synthesis, andquantification. The use of a standardized control RNA allows moreefficient assays, quantification, and comparative testing.

Another aspect of the invention includes a device for performing highthroughput quantification of mRNA in whole blood, wherein the deviceincludes: (a) a multi-well plate containing: a plurality ofsample-delivery wells; a leukocyte-capturing filter underneath thewells; and an mRNA capture zone underneath the filter which containsimmobilized oligo(dT); and (b) a vacuum box adapted to receive thefilter plate to create a seal between the plate and the box. In onepreferred embodiment of the device, the leukocytes are captured on aplurality of filter membranes that are layered together. In anotherpreferred embodiment of the device, the vacuum box is adapted to receivea source of vacuum. In another preferred embodiment of the device, amulti-well supporter is inserted between the vacuum box and themulti-well plates.

Another aspect of the invention includes a kit, which contains: thedevice for performing high throughput quantification of mRNA in wholeblood, heparin, a hypotonic buffer, and a lysis buffer.

Another aspect of the invention includes a fully automated system forperforming high throughput quantification of mRNA in whole blood,including: a robot to apply blood samples, hypotonic buffer, and lysisbuffer to the device; an automated vacuum aspirator and centrifuge, andautomated PCR machinery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded drawing of the high throughput mRNA device.

FIG. 2 depicts the multi-well plate, including the leukocyte filter andoligo-(dT)-immobilized filter wells, of the high throughput mRNA device.

FIG. 3 is a graph showing the efficiency of leukocyte trapping of freshand frozen blood samples on filter plates.

FIG. 4 is a graph showing the effect of number of washes of blood onmRNA quantification.

FIG. 5 is a graph showing the effect of final treatments of filterplates before cell lysis on mRNA quantification.

FIG. 6 is a graph showing how lysis buffer inhibits RNase.

FIG. 7 is a graph showing optimal concentrations of reversetranscriptase for mRNA quantification.

FIG. 8 is a graph showing optimal values of cDNA for PCR to capturemRNA.

FIG. 9 is a graph showing the hybridization kinetics of the invention.

FIG. 10 is a graph showing the linear relationship between whole bloodvolume used per well and mRNA quantification.

FIG. 11 is a graph showing optimal guanidine thiocyanate concentration.

FIG. 12 is a graph showing optimal proteinase K concentration.

FIGS. 13A-13D are graphs showing assay validation.

FIGS. 14A-14D are graphs showing recovery of synthetic spiked RNA.

FIG. 15. shows cDNA synthesis from a specific antisense primer (NNN) andimmobilized oligo(dT).

FIG. 16. is a graph showing recovery of specifically primed RNA with andwithout denaturization.

FIG. 17. is a graph showing amplification of RNA with and withoutspecific primers.

FIG. 18A shows a typical mRNA capture scheme of the present invention.

FIG. 18B is a graph showing various hybridization performances.

FIG. 18C is a graph showing efficiency of various methods of capturingtotal RNA and Poly(A) RNA.

FIG. 18D is a graph showing the optimal number of PCR cycles.

FIG. 18E is a graph showing the optimal range of lysis buffer forcapture of RNA.

FIG. 19A is a graph showing number of PCR cycles versus variousanticoagulants.

FIG. 19B is a graph showing number of PCR cycles versus storage time.

FIG. 19C is a graph showing number of PCR cycles versus hybridizationtemperature.

FIG. 19D is a graph showing number of PCR cycles versus hybridizationtime.

FIG. 19E is a graph showing number of PCR cycles versus units of MMLV.

FIG. 19F is a graph showing number of PCR cycles versus μl of cDNA perwell.

FIG. 20A is a graph showing Ct values for spiked standard RNA.

FIG. 20B is a graph showing percent recovery for spiked standard RNA.

FIG. 20C is a graph showing Ct values for inhibitor dA20.

FIG. 20D is a graph showing percent inhibition for inhibitor dA20.

FIG. 20E is a graph showing Ct values per μl of blood.

FIG. 20F is a graph showing mRNA recovery per μl of blood.

FIG. 21A is a graph showing percent recovery of standard RNA amongvarious subjects.

FIG. 21B is a graph showing CD4 mRNA per μl of blood recovered amongvarious subjects.

FIG. 21C is a graph showing p21 mRNA per μl of blood recovered amongvarious subjects.

FIG. 21D is a graph showing FasL mRNA per μl of blood recovered amongvarious subjects.

FIG. 21E is a graph showing LTC4S mRNA per μl of blood recovered amongvarious subjects.

In FIGS. 22A-H, Δ shows p21 mRNA for the control stimulation, ▴ showsp21 mRNA for the PMA+CaI stimulation, ⋄ shows FasL mRNA for the controlstimulation, and ♦ shows FasL mRNA for the PMA+CaI stimulation.

FIG. 22A is a graph showing in vitro induction of mRNA in whole blood.

FIG. 22B is a graph showing blood storage before in vitro stimulation.

FIG. 22C is a graph showing in vitro induction of FasL mRNA amongvarious subjects, by showing mRNA molecules/mL blood for bothstimulation and vehicle control, respectively.

FIG. 22D is a graph showing in vitro induction of p21 mRNA among varioussubjects, by showing mRNA molecules/mL blood for both stimulation andvehicle control, respectively.

FIG. 22E is the same graph as FIG. 22C rotated until the regression linebecomes horizontal.

FIG. 22F is the same graph as FIG. 22D rotated until the regression linebecomes horizontal.

FIG. 22G graphs the same data as FIG. 22C, showing fold increase forindividual subjects.

FIG. 22H graphs the same data as FIG. 22D, showing fold increase forindividual subjects.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention allows analysis of larger volumes of unpreparedwhole blood, provides an efficient means of analyzing mRNA that isderived exclusively from white blood cells; removes rRNA and tRNA,provides consistent mRNA recovery, and is easily adaptable toautomation. The present invention provides a sensitive quantificationsystem, including: absolute quantification using real time PCR, andexcellent reproducibility with coefficients of variation ranging from20-25%. Moreover, the present invention is applicable to various diseasetargets (Table I).

TABLE I Clinical targets Stimulation Diseases Candidate Genes (−)Leukemia Translocation gene Cancer (diagnostics, Cancer-specific genefrom monitoring, screening) micrometastatic cancer cells HIV/CMV(diagnostics, Virus-derived mRNA monitoring, blood bank) from infectedWBCs In vivo Anti-leukemia drugs Apoptosis sensitivityImmuno-suppressant Cytokines Side effect of anti-cancer Housekeepinggenes drugs on WBCs In vitro Anti-viral drug sensitivity Virus-derivedmRNA from infected WBCs

The invention is not limited to any particular mechanical structure.However, FIGS. 1 and 2 show a preferred structure for implementing thehigh throughput mRNA quantification of the present invention. A vacuumbox 10 forms the base of the structure. The vacuum box can be made ofany material sufficiently strong to withstand vacuum aspiration;however, disposable plastic material is preferred. The vacuum box isadapted to receive a source of vacuum in order to perform vacuumaspiration 12. A filter plug 14 is located within the vacuum aspiratoradapter of the vacuum box. The vacuum box 10 preferably has a ledge 16to mate with a multi-well filter plate 40, or optionally, a multi-wellsupporter 20. The multi-well supporter 20 is optionally provided insidethe upper part of the vacuum box so as to support the multi-wellfilterplate 40. A sealing gasket 30, preferably comprised ofsilicon-based rubber or other soft plastic, is located on top of themulti-well supporter. Above the sealing gasket lies the multi-wellfilter plate 40, which contains multiple sample wells 46, multipleleukocyte-capturing filters 42 underneath the sample-delivery wells, andan mRNA capture zone 44 under the filter. Oligo(dT)-immobilized iscontained in the wells of the mRNA capture zone.

One preferred embodiment involves a simple, reproducible, and highthroughput method of mRNA quantification from whole blood. The rapidprotocol minimizes the secondary induction or degradation of mRNA afterblood draw, and the use of 96-well filterplates and microplates allowsthe simultaneous manipulation of 96 samples. Minimal manipulation duringthe procedure provides for very small sample-to-sample variation, withcoefficient of variation (CV) values of less than 30%, even when PCR isused as a means of quantification.

In one embodiment, the method involves preparation of the vacuum box. Inone preferred embodiment, a blood encapsulator such as polyacrylatepolymer matrix (Red Z, Safetec) is added to the vacuum box to solidifythe blood. A multi-well supporter is then placed in the vacuum box. Asealing gasket made of silicon-based rubber or other soft plastics isthen placed on top of the multi-well plate supporter. A filter plug(X-6953, 60μ Filter Plug HDPE, Porex Products Groups) is placed in thevacuum aspirator adapter of the vacuum box.

In this embodiment, the method involves the preparation of the filterplate. Either glassfiber membranes or leukocyte filter membranes can beused to capture leukocytes. In order to simplify the assay,multiple-well filterplates are constructed using glassfiber membranes orleukocyte filter membranes to enable the simultaneous processing ofmultiple blood specimens. Examples of filters for capturing leukocytesare disclosed in U.S. Pat. Nos. 4,925,572 and 4,880,548, the disclosuresof which are hereby incorporated by reference. Adsorption of leukocyteson fiber surfaces is generally accepted as the mechanism of leukocyteremoval. Since the surface area of a given weight of fibers is inverselyproportional to the diameter of the fibers, it is to be expected thatfiner fibers will have higher capacity and that the quantity as measuredby weight of fibers necessary to achieve a desired efficiently will beless if the fibers used are smaller in diameter. A number of commonlyused fibers, including polyesters, polyamides, and acrylics, lendthemselves to radiation grafting, as they have adequate resistance todegradation by γ-radiation at the levels required for grafting and areof a structure with which available monomers can react. PBT has been theprincipal resin used for the development of the products of thisinvention and is the resin used in the examples. It should be noted,however, that other resins may be found which can be fiberized andcollected as mats or webs with fibers as small as 1.5 micrometers orless, and that such products, with their critical wetting surfacetensions adjusted as necessary to the optimum range, may be well suitedto the fabrication of equally efficient but still smaller leukocytedepletion devices. Similarly, glass fibers, appropriately treated, maybe usable to make effective devices. Absorption of CD4 mRNA is up tofour times as effective when using PBT-based filters as opposed to glassfiber-based filters. The filter plate is placed in the vacuum box. Inanother preferred embodiment, multiple filter membranes are layeredtogether to increase the amount of leukocytes captured from whole blood.In one preferred embodiment, the filter plate is placed upon the platesupporter and the sealing gasket. In another preferred embodiment, thefilter plate is sealed with a plastic adhesive tape (Bio-Rad 223-9444),and the tape is cut to allow access to a desired number of wells. Inanother preferred embodiment, each well to which a sample will be addedis washed with a hypotonic buffer (200 μL 5 mM Tris, pH 7.4).

The method preferably involves collecting blood, adding the blood to themulti-well filter plate, and removal of erythrocytes and othernon-leukocyte components. In one preferred embodiment, whole blood canbe drawn into blood collection tubes containing anticoagulants, whichincrease the efficiency of the leukocyte filtering. The anticoagulant,heparin, is particularly effective in increasing the efficiency ofleukocyte filtering. In one preferred embodiment, the blood sample canbe frozen, which removes some of the RNAases that destroy mRNA. Thewells can be washed with a hypotonic buffer. Once blood has been addedto the desired number of wells on the filterplate, the blood is filteredthrough the filter membrane. Filtration can be affected through anytechnique known to those of skill in the art, such as centrifugation,vacuum aspiration, or positive pressure.

In one especially preferred embodiment, vacuum aspiration is commenced(with 6 cm Hg) after the blood samples have been added to thefilterplate wells. Each well is washed several times with a hypotonicbuffer (12× with 200 μL 5 mM Tris, pH 7.4). In another preferredembodiment, each well containing a sample is washed with ethanol (1×with 200 μL 100% ethanol), which dries the filter membrane andsignificantly increases the efficiency of leukocyte trapping duringvacuum aspiration. In another preferred embodiment, the vacuum is thenapplied (20 cm Hg for >2 min).

The method involves cell lysis and hybridization of mRNA to theoligo(dT)-immobilized within the mRNA capture zone. Lysis buffer isapplied to the filterplate wells (40 μL/well), and incubation is allowedto occur (room temperature for 20 min) to release mRNA from the trappedleukocytes. In one preferred embodiment, the multi-well filterplate issealed in a plastic bag and centrifuged (IEC MultiRF, 2000 rpm, at 4 C,for 1 min). Lysis buffer is then added again (20 μL/well), followed bycentrifugation (IEC MultiRF, 3000 rpm, at 4 C, for 5 min). Themulti-well filterplate is then removed from the centrifuge and incubated(room temperature for 2 hrs).

In accordance with a preferred embodiment, the lysis buffer comprises adetergent, a salt, a pH buffer, guanidine thiocyanate, and proteinase K.

Preferred embodiments of the lysis buffer contain at least onedetergent, but may contain more than one detergent. Those skilled in theart may utilize different combinations of concentrations of detergentswith different strengths in order to achieve varying levels of lysis ofdifferent membranes for various types of cells. For example, IGEPALCA-630 is a weaker detergent than N-laurosarcosine, and in oneembodiment IGEPAL CA-630 alone may be sufficient to lyse a cytoplasmicmembrane. In other embodiments, a strong detergent, such asN-laurosarcosine can be used in combination with one or more weakdetergents to optimize lysis of nuclear membranes. The detergents arepreferably sufficient to lyse at least the cytoplasmic membrane ofcells. Another preferred embodiment comprises a detergent sufficient tolyse the nuclear membrane of cells, as significant amounts of mRNAreside in the nuclei of cells. In some circumstances it is desirable tomeasure only cytoplasmic mRNA, while in other circumstances, it may bedesirable to measure mRNA in the cytoplasm and nucleus.

Strong detergents of the lysis buffer preferably include, but are notlimited to: N-lauroylsarcosine, S.D.S., Sodium deoxycholate, andHexadecyltrimethylammonium bromide.

Weak detergents include IGEPAL CA-630, N-Decanoyl-N-methylglucamine,Octyl-β-D-glucopyranoside, or other detergents known to those skilled inthe art. 0.05-2% detergent can be used in the lysis buffer. Oneparticularly preferred embodiment of the lysis buffer includes 0.5%N-lauroylsarcosine. Another preferred embodiment of the lysis buffercontains 0.1-2% IGEPAL CA-630. A particularly preferred embodimentcontains 0.1% IGEPAL CA-630.

The combination of salts and chelating agents can also serve as a lysingagents. For example, 75 μM NaCl and 24 μM Na-EDTA can serve as a lysingagent. Embodiments of lysing agents may include other lysing agentsknown to those skilled in the art.

The salt of the lysis buffer acts as an mRNA-oligo(dT) hybridizingagent. The salt should preferably have a stringency (the rigor withwhich complementary DNA sequences hybridize together) that does notexceed that of 4×SSC, as determinable by those skilled in the art. Otherembodiments of the lysis buffer include NaCl or other salts known tothose skilled in the art.

The pH buffer of the lysis buffer stock preferably maintains a pH of7.0-8.0. One embodiment comprises 1 mM-100 mM Tris HCl, pH 7.4. In aparticularly preferred embodiment, the pH buffer comprises 10 mM TrisHCl, pH 7.4. Other preferred embodiments of the lysis buffer include pHbuffers known to those skilled in the art, including 0.1 MCitrate-Phosphate, pH 5.0, with 0.03% H₂O₂.

In accordance with a particularly preferred embodiment of the lysisbuffer, guanidine thiocyanate serves as an RNAase deactivating agent. Wehave discovered that guanidine thiocyanate has typically been used inthe prior art at insufficient concentrations to be effective. Therefore,preferably, the concentration of guanidine thiocyanate is greater than1.4 M. Guanidine thiocyanate concentration as high as 10 M, morepreferably no higher than 2 M can be used. However, as seen in FIG. 11,at concentrations above 1.7 M, the efficiency of the lysis buffer isdecreased. Accordingly, the preferred embodiment uses about 1.4 to about1.75 M guanidine thiocyanate. One preferred embodiment comprises 1.7-1.8M guanidine thiocyanate. A working lysis buffer can be prepared from thestock, as demonstrated in Example 4 below, with the particularconcentration of 1.791 M guanidine thiocyanate. As other reagents areadded to the lysis buffer, the concentration of guanidine thiocyanatebecomes diluted. Where 55 ml of other reagents are added to 1 ml of thebuffer as in Example 4, the preferred lysis buffer comprises guanidinethiocyanate in concentrations of about 1.61 to about 1.71 M. Thus, apreferred embodiment comprises guanidine thiocyanate in concentrationsof about 1.6 to about 1.7 M.

A particularly preferred embodiment further comprises 20 mg/ml ofproteinase K as an RNAase inactivating agent. One preferred embodimentof the lysis buffer comprises 200 μg/ml-20 mg/ml of proteinase K.Another preferred embodiment comprises 200 μg/ml-1.0 mg/ml proteinase K.Another preferred embodiment comprises 200 μg/ml-500 μg/ml proteinase K.Sodium dodecyl sulfate may also serve as the RNAase deactivating agent.Another embodiment includes 0.1-10% of 2-mercaptoethanol as an RNAaseinactivating agent. One particularly preferred embodiment comprises 1%2-mercaptoethanol. Other embodiments of RNAase inactivating agents maypreferably include materials, known to those skilled in the art, thatreduce disulfide bonds in RNAases.

Preferred embodiments of the lysis buffer further comprise chelatingagents which chelate Mg²⁺ and Ca²⁺. One preferred embodiment comprises0.1 mM-5 mM EDTA. A particularly preferred embodiment comprises 1 mMEDTA. Other preferred embodiments of the lysis buffer stock containchelating agents known to those skilled in the art including, forexample and without limitation, EDTMP, 2,3-dimercaptopropanol, and EGTA.

Preferred embodiments of the lysis buffer may include tRNA, which maycome from various sources and is included in order to inhibitnon-specific absorption of blood-derived DNA and RNA to filter plates.Additionally, the presence of tRNA prevents degradation of blood-derivedRNA. In one preferred embodiment, the tRNA of the working lysis buffercomprises 10 mg/ml of E. coli tRNA. Other embodiments may contain tRNAfrom any source known to those skilled in the art.

Preferred embodiments of the lysis buffer may include DNA from a widevariety of sources, which is added in order to inhibit non-specificabsorption of blood-derived DNA and RNA to filter plates. The DNA of theworking lysis buffer preferably comprises 10 mg/ml of sonicated salmonsperm DNA. In other embodiments, DNA from other organisms may be used.

Particularly preferred embodiments of the lysis buffer may includespiked control RNA to calculate the definite quantity of target mRNAs inthe original samples. Prior to embodiments of the present invention, itwas difficult to compare the results in one experiment to those in otherexperiments due to institute-to-institute variation and lack ofstandardization. However, in preferred embodiments of the presentinvention a definite quantity of target mRNA can be determined bydividing the values obtained by the TaqMan assay with percent recoveryof a dose of spiked control RNA in each sample. Such definitivequantification is described below and exemplified in Example 5.

Preferred embodiments of the lysis buffer include 10 to 1e¹⁰, morepreferably 1e⁵ to 1e¹⁰, copies of spiked RNA per well. In preferredembodiments, the amount of control RNA used is at least enough to bedetected, but not so much as to significantly interfere with the amountof target mRNA that is quantified. In preferred embodiments, the controlRNA added to the lysis buffer is poly(A)⁺ RNA. In particularly preferredembodiments where the sample being tested is human blood, the controlRNA is not homologous to RNA present in human blood. In some preferredembodiments, the sequence of the control RNA is less than 90% homologousto the target mRNA, or has greater than 10% difference in length withthe target mRNA. In other preferred embodiments, the sequence of thecontrol RNA is less than 85% homologous to the target mRNA, or hasgreater than 5% difference in length with the target mRNA. In furtherembodiments, the sequence of the control RNA is less than 75% homologousto the target mRNA, or has greater than 2% difference in length with thetarget mRNA. In alternative embodiments, the sequence of the control RNAis less than 65% homologous to the target mRNA, or has greater than 1%difference in length with the target mRNA. In one embodiment, controlRNA may preferably be made by amplifying template oligonucleotides bymeans of PCR. Thus, forward primers (SEQ ID NOs 10, 11, 15, and 8),reverse primers (SEQ ID NOs 9 and 16), and TaqMan probes (SEQ ID NOs 13,17, and 12) can be used to amplify various control RNA oligonucleotides.Alternative embodiments comprise using a plurality of different targetmRNAs to be quantified. Further embodiments comprise using a pluralityof control RNAs.

The method involves quantification of mRNA, which in a preferredembodiment entails cDNA synthesis from mRNA and amplification of cDNAusing PCR. In one preferred embodiment, the multi-well filterplate iswashed with lysis buffer (150 μL/well×3 times, manual) and wash buffer(150 μL/well×3 times, manual or BioTek #G4). A cDNA synthesis buffer isthen added to the multi-well filterplate (40 μL/well, manual or I&J #6).Axymat (Amgen AM-96-PCR-RD) can be placed on the multi-well filterplate,which is then placed on a heat block (37 C, VWR) and incubated (>90min). The multi-well filterplate can then be centrifuged (2000 rpm, at 4C for 1 min). PCR primers are added to a 384 well PCR plate, and thecDNA is transferred from the multi-well filterplate to the 384 well PCRplate. The PCR plate is centrifuged (2000 rpm, at 4 C for 1 min), andreal time PCR is commenced (TaqMan/SYBER).

Another preferred embodiment comprises application of specific antisenseprimers during mRNA hybridization or during cDNA synthesis, asdemonstrated in Example 6 below. The oligo(dT) and the specific primer(NNNN) simultaneously prime cDNA synthesis at different locations on thepoly-A RNA (FIG. 15). The specific primer (NNNN) and oligo(dT) cause theformation of cDNA during amplification, as shown in FIG. 15. Even whenthe specific primer-derived cDNA is removed from the GenePlate byheating each well at 95 degrees C. for two minutes, the amounts ofspecific CD4 cDNA obtained from the heat denaturing process (usingTaqMan quantitative PCR) is similar to the amount obtained from anun-heated negative control (FIG. 16). Without wishing to be bound by anyexplanation or theory, one possible explanation for such results is thatoligo(dT)-derived cDNA may displace primer-derived cDNA duringamplification (FIG. 15). This is particularly convenient because theheat denaturing process is completely eliminated. Moreover, by addingmultiple antisense primers for different targets, each gene can beamplified from the aliquot of cDNA, and oligo(dT)-derived cDNA in theGenePlate can be stored for future use.

Another preferred embodiment of the invention involves a device forhigh-throughput quantification of mRNA from whole blood. The deviceincludes a multi-well filterplate containing: multiple sample-deliverywells, a leukocyte-capturing filter underneath the sample-deliverywells, and an mRNA capture zone under the filter, which containsoligo(dT)-immobilized in the wells of the mRNA capture zone. In order toincrease the efficiency of leukocyte collection, several filtrationmembranes can be layered together. The multi-well plate is fitted upon avacuum box, which is adapted to receive the plate and to create a sealbetween the multi-well plate and the vacuum box. In one preferredembodiment of the device, the vacuum box is adapted to receive a sourceof vacuum in order to perform vacuum aspiration. In another preferredembodiment, a multi-well supporter is placed in the vacuum box, belowthe multi-well filterplate. In another preferred embodiment of thedevice, a sealing gasket, which can be made from soft plastic such assilicon-based rubber, is inserted between the multi-well supporter andthe multi-well filterplate.

Although many conventional amplification techniques can be used inconjunction with the present invention, one particularly preferredembodiment of the present invention comprises conducting real-timequantitative PCR (TaqMan) with whole blood-derived RNA and control RNA.Holland, et al., PNAS 88:7276-7280 (1991) describe an assay known as aTaqman assay. The 5′ to 3′ exonuclease activity of Taq polymerase isemployed in a polymerase chain reaction product detection system togenerate a specific detectable signal concomitantly with amplification.An oligonucleotide probe, nonextendable at the 3′ end, labeled at the 5′end, and designed to hybridize within the target sequence, is introducedinto the polymerase chain reaction assay. Annealing of the probe to oneof the polymerase chain reaction product strands during the course ofamplification generates a substrate suitable for exonuclease activity.During amplification, the 5′ to 3′ exonuclease activity of Taqpolymerase degrades the probe into smaller fragments that can bedifferentiated from undegraded probe. The assay is sensitive andspecific and is a significant improvement over more cumbersome detectionmethods. A version of this assay is also described in Gelfand et al., inU.S. Pat. No. 5,210,015. U.S. Pat. No. 5,210,015 to Gelfand, et al., andHolland, et al., PNAS 88:7276-7280 (1991), which are hereby incorporatedby reference.

Further, U.S. Pat. No. 5,491,063 to Fisher, et al., provides aTaqman-type assay. The method of Fisher et al. provides a reaction thatresults in the cleavage of single-stranded oligonucleotide probeslabeled with a light-emitting label wherein the reaction is carried outin the presence of a DNA binding compound that interacts with the labelto modify the light emission of the label. The method utilizes thechange in light emission of the labeled probe that results fromdegradation of the probe. The methods are applicable in general toassays that utilize a reaction that results in cleavage ofoligonucleotide probes, and in particular, to homogeneousamplification/detection assays where hybridized probe is cleavedconcomitant with primer extension. A homogeneous amplification/detectionassay is provided which allows the simultaneous detection of theaccumulation of amplified target and the sequence-specific detection ofthe target sequence. U.S. Pat. No. 5,491,063 to Fisher, et al. is herebyincorporated by reference.

The TaqMan detection assays offer several advantages over the classicalPCR assays. First, the TaqMan assays combine the sensitivity of PCRalong with hybridization of the internal oligonucleotide sequence thatis present in a target sequence. Following PCR, samples do not have tobe separated on agarose gels, and the subsequent Southern blots andhybridization steps that are necessary to verify the identity of the PCRproducts is eliminated. These additional post-PCR confirmation steps caneasily add several days for an accurate identification. Using the TaqMansystem, the assays are completed within 2.5 h. Further, the methodologyinvolved in the assay process makes possible the handling of largenumbers of samples efficiently and without cross-contamination and istherefore adaptable for robotic sampling. As a result, large numbers oftest samples can be processed in a very short period of time using theTaqMan assay. Another advantage of the TaqMan system is the potentialfor multiplexing. Since different fluorescent reporter dyes can be usedto construct probes, several different HIV systems could be combined inthe same PCR reaction, thereby reducing the labor costs that would beincurred if each of the tests were performed individually. Theadvantages of rapid, conclusive data together with labor and costefficiency make the TaqMan detection system utilizing the specificprimers of the invention a highly beneficial system for monitoring thepresence of HIV.

In preferred embodiments, various mRNAs can be quantitated by simplychanging primers and probes for each target. Because heparin maintainsextracellular Ca⁺⁺, which is one of important factors to exert maximumbiological activities, drug actions can be analyzed in whole bloodwithout isolating leukocytes.

In preferred embodiments of the present invention, the ability todetermine the total efficiency of a given sample by using known amountsof spiked standard RNA results from embodiments being dose-independentand sequence-independent. The use of known amounts of control RNA allowsPCR measurements to be converted into the quantity of target mRNAs inthe original samples. Such calculations can be used on large samples ofindividuals over time in order to determine normal ranges of presence ofmRNA per μl of whole blood for various genes. When embodiments of thepresent invention are used to test individuals' whole blood at a giventime, results indicating the presence of mRNA indicative of disease,with levels falling outside of the normal ranges, may signal thepresence of disease. Embodiments of the present invention can be used asassays for the detection of various diseases. For example, mRNAindicative of a disease can be detected by inducing mRNA in many samplesto a maximum desired level, measuring the mRNA of a given sample, anddetecting the level of mRNA of the sample to determine if it falls belowthe maximum level. Similarly, embodiments of the present invention canbe used as assays in determining the effectiveness of a therapeuticregimen. Embodiments of the present invention can similarly be used inoxidative stress tests, where mRNA levels of samples of people usingvarying amounts of anti-oxidants are compared to each other.

Another preferred embodiment involves a kit for high-throughputquantification of mRNA from whole blood. The kit includes: the devicefor high-throughput quantification of mRNA from whole blood;heparin-containing blood-collection tubes; a hypotonic buffer; and alysis buffer.

Another preferred embodiment involves a fully automated system forperforming high throughput quantification of mRNA in whole blood,including: robots to apply blood samples, hypotonic buffer, and lysisbuffer to the device; an automated vacuum aspirator and centrifuge, andautomated PCR machinery.

EXAMPLES Example 1

Various protocols of the method of the present invention were tested andused to quantify β-actin mRNA and CD4 mRNA from whole blood.

Three anticoagulants were tested: ACD, EDTA, and heparin, with heparinresulting in the highest percent of leukocyte retention. While Leukosorbmembranes have been used for ACD blood in transfusion, approximately15-40% of leukocytes passed through even when four layers of membraneswere simultaneously used. EDTA blood was tested; the capacity andleukocyte retention was found to be similar to those for ACD. Mostnotably, however, was that 100% of the leukocytes in heparin blood weretrapped on the Leukosorb membranes. The capture of 100% of leukocytesfrom heparin blood shows the reliability of quantification of mRNA usingthe present invention. These data indicate that the use of heparin bloodis most suitable for the precise quantification of mRNA, whereas ACDblood is useful for applications requiring larger volumes of blood andless quantitative results.

The results of using of frozen versus fresh blood samples were compared.As indicated in FIG. 3, more CD4 mRNA was recovered from leaked cells offresh blood than from leaked cells of frozen samples.

The effectiveness of whole blood retention of glassfiber filters, ascompared to retention values of PBT-based filter membranes, was alsoexamined. As shown in Table II, glassfiber membranes accepted only 40 μLof whole blood, even when membranes were washed with hypotonic buffer(50 mM Tris, pH 7.4) to burst erythrocytes. Leukosorb filters, however,accepted significantly larger amounts of whole blood than the glassfiberfilters, as indicated in Table II.

TABLE II Amplification of β-actin mRNA From Whole Blood Maximum Testedblood WBC blood volume, retention Subject volume, MembraneAnticoagulant*¹ μl/well (%) (n) μl/well ×10⁻⁵ ng (CV⁺⁵, %) CT*³ (CV*⁴,%) Glass fiber A, E, H 40 100 #1 (9) 20 (A)*² 4.84 ± 2.80 (57.9) 38.0 ±0.876 (2.31) #2 (9) 20 (A) 4.02 ± 2.52 (62.7) 38.4 ± 0.994 (2.59)Leukotrap A, E 3000 60-85 #1 (9) 1000 (A) 169 ± 112 (66.0) 33.1 ± 1.24(3.75) #2 (9) 1000 (A)  100 ± 72.1 (72.0) 30.6 ± 1.42 (4.64) H 200 100#1 (9) 100 (H) 18.7 ± 4.03 (21.6) 39.2 ± 0.414 (1.01) #2 (9) 100 (H)15.7 ± 3.67 (23.4) 37.8 ± 0.432 (1.06) #3 (9) 100 (H) 15.8 ± 3.91 (24.7)37.7 ± 0.379 (1.01) #4 (9) 100 (H) 15.3 ± 4.39 (28.7) 37.7 ± 0.429(1.14) #5 (9) 100 (H) 13.5 ± 3.31 (24.5) 37.9 ± 0.455 (1.20) #6 (9) 100(H) 20.1 ± 4.79 (23.8) 37.3 ± 0.365 (0.98) *¹A: ACD, E: EDTA, H: heparin*²(A, H) represents anticoagulants used in the experiments. *³(CT:Threshold Cycle *⁴CV: Coefficient of variation

Various numbers of washes with hypotonic buffer were applied to removeerythrocytes and other blood components. As indicated in FIG. 4, washingthe samples with hypotonic buffer at least three times more than doubledthe amount of CD4 mRNA that was captured as compared to no washing. FIG.4 also shows that washing the blood twelve times with hypotonic bufferresulted in the capture of the most mRNA. Additionally, various methodsof vacuuming, centrifuging, and washing with ethanol followed byvacuuming blood samples to collect leukocytes were compared with respectto final CD4 mRNA quantification. FIG. 5 indicates that while vacuumaspiration resulted in better CD4 mRNA quantification thancentrifugation, washing blood samples with ethanol prior to vacuumaspiration yields the most mRNA.

Once the leukocytes were trapped on the glassfiber or Leukosorbmembranes, various numbers of washes with hypotonic buffer were appliedto remove erythrocytes and other blood components. To release mRNA fromthe trapped leukocytes, lysis buffer (RNAture) was applied to thefilterplates (40 μL/Well), and the plates were incubated at roomtemperature for 20 minutes. In a preferred embodiment, amplificationprimers are included in the lysis buffer. FIG. 6 indicates that lysisbuffer plays an important role in RNAase inhibition; eosinophils arefilled with RNAases, which are inactivated by the lysis buffer. Themulti-well filterplate was then sealed in a plastic bag and centrifuged(IEC MultiRF, 2000 rpm, at 4 C, for 1 min). Lysis buffer was then addedagain (20 μL/well), followed by centrifugation (IEC MultiRF, 3000 rpm,at 4 C, for 5 min). The multi-well filterplate was then removed from thecentrifuge and incubated at room temperature for two hours to allowhybridization of poly(A)+ RNA tails with the immobilized oligo(dT). Themulti-well filterplates were then washed three times with 150 μL LysisBuffer to remove remaining ribonucleases, followed by three washes with150 μL Wash Buffer (BioTek #G4) to remove the Lysis Buffer, whichcontained some inhibitors of cDNA synthesis.

Upon the final wash, the Wash Buffer was completely removed from themulti-well filterplates, and cDNA was synthesized in each well by adding40 μL of premixed cDNA buffer. The cDNA buffer preferably consists of:5× First Strand Buffer (Promega M531A, 10 mM dNTP (Promega stock, 20×)),Primer (5 μM, #24), RNasin (Promega N211A, 40 U/μL), M-MLV reversetranscriptase (Promega M170A, 200 U/μL), and DEPC water. FIG. 7indicates that the optimal concentration of MMLV for mRNA quantificationis 50 units/well.

Axymat (Amgen AM-96-PCR-RD) was placed on the multi-well filterplate,which was then placed on a heat block (37 C, VWR) and incubated (>90min). The multi-well filterplate was then centrifuged (2000 rpm, at 4 Cfor 1 min). PCR primers were added to a 384-well PCR plate, and the cDNAwas transferred from the multi-well filterplate to the 384-well PCRplate. FIG. 8 indicates that the optimal value of cDNA for PCR isapproximately 2 μL/well. The PCR plate was centrifuged (2000 rpm, at 4 Cfor 1 min), and real time PCR was commenced (TaqMan/SYBER). The methodof the current invention has high mRNA specificity; amplification of CD4mRNA with TaqMan qPCR resulted in undetectable DNA contamination (<10copies/well).

As indicated in FIG. 9, the present invention results in lowcoefficients of variation for mRNA quantification. Hybridization for twohours resulted in a coefficient of variation of less than 13%, ascompared to traditional coefficients of variation of approximately 300%for mRNA quantification. Moreover, as indicated in FIG. 10, the linearresults show that the amount of mRNA that is captured is directlyproportional to the volume of whole blood used per well, making thepresent invention a reliable and reproducible method of quantifyingmRNA.

Example 2

Fifty μL of heparinized frozen human blood was applied to the Leukosorbfilterplate. Each well was vacuumed and washed twelve times with 150 μLof 5 mM Tris pH 7.4 and 150 μL of 100% ethanol. Then, 40 μL of lysisbuffer, which contains 1.707-1.856 M guanidine thiocyanate, was added tothe well. After incubation at room temperature for 15 min, thefilterplate was placed onto the GenePlate and centrifuged at 2000 rpm at4° C. for 1 min. An additional 20 μL Lysis Buffer was added and thesample centrifuged for 5 minutes. After the GenePlate was incubated atroom temperature for 2 hours, each well was washed with 100 μL LysisBuffer 3 times, followed by three applications of 150 μL Wash Buffer (10mM Tris, pH 7.4, 1 mM EDTA, pH 8.0, 0.5 M NaCl). The cDNA wassynthesized in the GenePlate, and 2 μL cDNA was used for the TaqManassay to quantitate CD4. The results are indicated in FIG. 11.

Example 3

Fifty μL of heparinized frozen human blood was applied to the Leukosorbfilterplate. Each well was vacuumed and washed twelve times 150 μL of 5mM Tris pH 7.4, and 150 μL of 100% ethanol. Then, 40 μL of Lysis Buffer,which contains 1.791 M guanidine thiocyanate with 0-0.5 mg/ml proteinaseK, was added to the wells. After incubation at room temperature for 15min, the filterplate was placed onto the GenePlate and centrifuged with2000 rpm at 4° C. for 1 min. An additional 20 μL of lysis buffer wasadded and centrifuged again for 5 min. After the GenePlate was incubatedat room temperature for 2 hours, each well was washed with 100 μL LysisBuffer 3 times, followed by 150 μL Wash Buffer (10 mM Tris, pH 7.4, 1 mMEDTA, pH 8.0, 0.5 M NaCl) 3 times. The cDNA was synthesized in theGenePlate, and 2 μL cDNA was used for TaqMan assay to quantitate CD4.The results are indicated in FIG. 12.

Example 4 Lysis Buffer Stock

0.5% N-Lauroylsarcosine

4×SSC

10 mM Tris HCl, pH 7.4

1 mM EDTA

0.1% IGEPAL CA-630

1.791 M guanidine thiocyanate

Working Lysis Buffer Lysis Buffer stock 1 ml 2-mercaptoethanol 10 μLSonicated salmon sperm DNA (10 mg/ml) 10 μL E. coli tRNA (10 mg/ml) 10μL Proteinase K (20 mg/ml stock) 25 μL (final 0.5 mg/ml)

Example 5

Preparation of control RNA. In order to synthesize control RNA, templateoligonucleotides (SEQ ID NOs 2 and 4) and cDNA from K562 cells (RNAture,Irvine, Calif.) were amplified with T7-forward primers (SEQ ID NOs 3, 5,and 6) and dT₄₀ reverse primers (SEQ ID NOs 1 and 7) with 30 cycles of95° C. denaturing for 30 sec, 55° C. annealing for 10 sec, followed by72° C. extension for 20 sec, respectively. Oligonucleotides werepurchased from IDT (Coralville, Iowa) or Proligo (Boulder, Colo.). Thesequences were as follows:

SEQ ID NO 1: 5′-T₄₀-GGGTG CTGTG CTTCT GTGAA C-3′, SEQ ID NO 2:5′-GCCCC CTCAC TCCCA AATTC CAAGG CCCAG CCCTC ACACATTGTT CACAG AAGCA CAGCA CCC-3′, SEQ ID NO 3:5′-GTAAT ACGAC TCACT ATAGG GGGAC AGCCC CCTCA CTCCC AAA-3′, SEQ ID NO 4:5′-GAAGC GTGTG TCACT GTGTG TTTCC AAGGC CCAGC CCTCACACAT TGTTC ACAGA AGCAC AGCAC CC-3′, SEQ ID NO 5:5′-GTAAT ACGAC TCACT ATAGG GGGAC GGAAG CGTGT GTCAC TGTGT GT-3′,SEQ ID NO 6: 5′-GTAAT ACGAC TCACT ATAGG GGGAC GCATT CCGCT GACCATCAAT A-3′, SEQ ID NO 7: T₄₀-TCCAA CGAGC GGCTT CAC-3′.

RNA was synthesized from purified PCR products by an in vitrotranscription system (T7 RiboMax Express, Promega) at 37° C. for 30 min,followed by 15 min of DNase (1 unit) treatment twice. Purified RNAproducts were suspended in nuclease-free water, and the concentrationswere determined by RiboGreen assay (Molecular Probes) with rRNA as astandard. The quality was analyzed by a capillary electrophoresis chip(iChip, Hitachi Chemical, Tokyo, Japan).

Leukocyte collection. Venous blood samples were collected from healthyadult volunteers. Glassfiber filterplates (RNAture) and Leukosorbmembranes (Pall Life Sciences, Ann Arbor, Mich.) were obtained from thedesignated suppliers. Custom 96-well Leukosorb filterplates weremanufactured by Whatman-Polyfiltronics (Clifton, N.J.). Since humanblood samples are considered contagious materials, a disposable vacuummanifold was designed, and custom products were manufactured by AmbrittEngineering (Santa Ana, Calif.). Filterplates were placed onto thedisposable vacuum manifold and washed twice with 200 μL of phosphatebuffered saline (PBS: Invitrogen, carlbad, CA). The vacuum was stopped,then fresh or thawed blood samples (up to 200 μL/well) were applied tothe filterplates. After all samples were dispensed into thefilterplates, vacuum filtration was started with 14 cm Hg, followed bywashing with PBS (200 μL/well) 12 times. After the final wash, thevacuum was continued for an additional 5 minutes to make the membranescompletely dry, and the residual volume of PBS was eliminated from themembranes.

Cell lysis and mRNA preparation. Filterplates were placed onto blankmicroplates, followed by application of 40 μL of lysis buffer (RNAture,Irvine, Calif.), which included a reverse primer (final concentrations25 nM), synthetic RNA as a quantitation standard, 100 μg/mL salmon spermDNA (Eppendorf-5 Prime, Westbury, N.Y.), 100 μg/mL E. coli tRNA (Sigma),500 μg/mL proteinase K (Pierce, Rockford, Ill.), and 1:100 dilution of2-mercaptoethanol (BioRad, Hercules, Calif.). The sample was incubatedat room temperature for 1 hour. In some experiments (FIGS. 14C-14D),various concentration of oligo dA₂₀ was added to the lysis buffer.Filterplates were then placed on oligo(dT)-immobilized microplates(GenePlate, RNAture), followed by centrifugation at 650×g for 1 min. 20μL of lysis buffer was then added, followed by centrifugation at 1450×gfor 5 minutes. After this process, the volume of lysis buffer in eachwell of the GenePlate was approximately 50 μL. After incubation of theGenePlate, the plate was washed with 100 μL plain lysis buffer 3 times,followed by washing with 150 μL wash buffer (10 mM Tris, pH 7.4, 1 mMEDTA, 0.5 M NaCl) 3 times.

cDNA synthesis. The cDNA was synthesized in a GenePlate by adding 30 μLof cDNA buffer, which contains 1×RT-buffer, 0.5 mM dNTP, 15 unitsrRNasin, and 37.5 units of MMLV reverse transcriptase (Promega). Thesample was incubated at 37° C. for 2 hours. Since reverse primers wereadded to the lysis buffer, primers were not included in the cDNAsynthesis reaction. After cDNA was synthesized, 50 μL of nuclease-freewater was added into each well, and 2 μL was used for TaqMan assay, asdescribed below.

TaqMan real time PCR. Primers and TaqMan probes for control RNA weredesigned by Primer Express version 2.0 (ABI, Foster City, Calif.). Forbcr-abl, we used published sequences. In some experiments, HYBsimulator(RNAture) was used to design reverse primers. The forward primers (SEQID NOs 10, 11, 15, and 8), reverse primers (SEQ ID NOs 9 and 16), andTaqMan probes (SEQ ID NOs 13, 17, and 12) were used to amplify controlRNA. In order to determine the amounts of CD4 mRNA in blood samples,both CD4 and control RNA were analyzed in the different wells of PCRplates, rather than multiplex PCR in a single well. For β-actin,commercially available primers and probes were used (ABI). Into a 384well PCR plate (ABI) were mixed: 2 μL of cDNA, 5 μL of TaqMan universalmaster mix (ABI), 1 μL of 5 μM of forward primers, 1 μL of 5 μM ofreverse primers, and 1 μL of 2 μM TaqMan probe. PCR was conducted in anABI PRISM 7900HT (ABI), using 1 cycle of 95° C. for 10 minutes, followedby 45 cycles of 95° C. for 20 seconds, followed by 55° C. for 20seconds, and finally 60° C. for 1 minute. The data were analyzed by SDSversion 2.0 (ABI). In some experiments, the TaqMan assay was conducteddirectly in a GenePlate (Opticon, MJ Research). Oligonucleotides (SEQ IDNOs 2, 4, and 14) and PCR products were used as quantitation standardsfor control RNA. The sequences were as follows:

SEQ ID NO 8: 5′-AAATG CCACA CGGCT CTCA-3′ SEQ ID NO 9:5′-CAAGT GTCTT CGTGT CGTGG G-3′ SEQ ID NO 10:5′-AGCCC CCTCA CTCCC AAA-3′ SEQ ID NO 11: 5′-AGCCC CCTCA CTCCC AAA-3′SEQ ID NO 12: 5′-FAM-CAGTG GCTAG TGGTG GGTAC TCAAT GTGTA CTT- TAMRA-3′SEQ ID NO 13: 5′-FAM-CCAAG GCCCA GCCCT CACAC A-TAMRA-3′ SEQ ID NO 14:5′-CAGG GACAA ATGCC ACACG GCTCT CACCA GTGGC TAGTGGTGGG TACTC AATGT GTACT TTTGG GTTCA CAGAA GCACA GCACC CAGGG-3′,SEQ ID NO 15: 5′-CCACT GGATT TAAGC AGAGT TCAA-3′ SEQ ID NO 16:5′-TCCAA CGAGC GGCTT CAC-3′ SEQ ID NO 17:5′-FAM-CAGCG GCCAG TAGCA TCTGA CTTTG A-TAMRA-3′

Data analysis. The amounts of PCR products were determined using thestandard curve for each gene. TaqMan results were then multiplied by thedilution factors (×40: 2 μL out of 80 μL of cDNA was used for PCR, and×1.67: 30 μL of cDNA was synthesized from 50 μL of lysis buffer/well).The percent recovery of the spiked RNA was further determined for eachsample by dividing recovered spiked RNA with pre-determined amounts ofspiked RNA (usually 10⁷ copies per well). For CD4 mRNA, TaqMan resultswere multiplied by the dilution factors, divided by blood volume, anddivided by percent recovery of the spiked RNA in the same sample. Eachblood sample was applied to 3 wells of filterplates (triplicate), andeach well produced a single cDNA and a single PCR for each gene.

Assay validation. Hybridization kinetics are shown in FIG. 13A, wherehybridization reached at plateau after 2 hours at room temperature.Blood dose dependency is demonstrated in FIG. 13B, where CD4 mRNA wasdetected at 0.05 μL, and increased linearly up to 200 μL in log scale.We also found that the hybridization efficiency was changed dramaticallybetween 15 and 25° C. (FIG. 13C). As shown in FIG. 13D, CD4 mRNA wasvery stable in heparinized blood and almost unchanged up to 7 hours at37° C. or overnight at 4° C. This suggests that CD4 may be a goodcontrol for gene expression analysis in whole blood. The results in FIG.13 were expressed as the mean±standard deviation of the number of genes.Although the CV of cycle threshold (Ct) was less than 1-2%, it increasedsubstantially when Ct was converted to the number of genes via thetransformation from log to linear scales. However, as shown in FIG. 13,the CV was as low as 10-30% even when the starting materials were wholeblood.

Quantitation. The goal of quantitation in this study was to determinethe total assay efficiency in each sample by using spiked control RNA,which is further used as a denominator to calculate the definitequantity of target mRNAs in the original samples. The principle includestwo assumptions: that the recovery of RNA is dose-independent, and thatrecovery is species-independent. In other words, percent recovery shouldbe identical between high and low copy number mRNAs, and also identicalamong different sequences. These assumptions are not only applied to thestep of mRNA purification, but also to whole processes of mRNAquantitation, from cell lysis to PCR. In order to validate the firstassumption, different amounts of synthetic poly(A)⁺ control RNA wereadded into the lysis buffer and exposed to Leukosorb membranes, where 50μL of blood was applied. After we confirmed that the control RNA was notamplified from human blood alone, the recovery of the control RNA wasdetermined by a TaqMan assay. As shown in FIG. 14A, dose-dependentrecovery of control RNA was observed at the tested range of 10⁵ to 10⁹copies/well. When the same data were converted to percent recovery,these values became similar around 20% (FIG. 14B). The data of FIGS. 14Aand 14B were the sum of whole processes, which include mRNA purificationand cDNA synthesis. Under the equilibrium condition of hybridization,the dissociation constant (Kd) was calculated as follows:

Kd=[RNA]×[oligo-dT]/[RNA:oligo-dT]; where [RNA] and [oligo-dT] representthe concentrations of unbound states of RNA and oligo-dT, respectively,and [RNA:oligo-dT] represents the concentrations of hybridized RNA witholigo-dT. This means that [RNA:oligo-dT] is variable of the amounts ofapplied RNA. In fact, [RNA:oligo-dT] was increased in proportion to theamounts of applied RNA, when hybridized RNA was measured by Yoyo-1nucleic acid dye (Miura, Y., Ichikawa, Y., Ishikawa, T., Ogura, M., deFries, R., Shimada, H., & Mitsuhashi, M. Fluorometric determination oftotal mRNA with oligo(dT) immobilized on microtiter plates. Clin Chem.42, 1758-64 (1996)). However, because the control RNA in FIG. 14B is avery small fraction of the total mRNA in each well, it does notpractically influence the values of Kd at this range. When primers wereincluded in the step of cDNA synthesis, we faced a problem of intra- andinter-species reproducibility. However, by adding primers duringhybridization, reproducibility improved, suggesting that cDNA is equallysynthesized from pre-hybridized primers, even when primer sequences aredifferent. Although percent recovery itself may vary among individualsdependent on the amounts of mRNA in samples, FIGS. 14A and 14B indicatethat percent recovery derived from one concentration can apply to otherconcentrations within the same samples.

To test the second assumption, that recovery is species-independent,hybridization was competitively inhibited by oligo-dA in lysis buffer,where three synthetic poly(A)⁺ RNAs were included. As shown in FIG. 14B,the amounts of recovered RNA varied among three spiked RNAs as well astarget native CD4 mRNA, because we intentionally used different amountsof RNAs. However, all RNAs were inhibited by oligo-dA at 3×10¹²-10¹⁵copies/well (FIG. 14C). Interestingly, when the same data weretransformed to the percent total, all four RNAs showed very similarinhibition curves with an IC₅₀ of around 3×10¹² copies/well (FIG. 14D).This suggests that mRNA purification in our system is poly(A)-specificand sequence-independent. As shown in FIG. 14C, some non-specificactivity remains even after 10¹⁵ copies of oligo-dA were applied.However, as shown in FIG. 14D, this non-specific activity was less than5% of total activity. Thus, non-poly(A) sequences do not appear to playa major role in this assay. Because FIGS. 14B and 14C were the sum ofwhole processes of mRNA quantitation, these graphs suggest that theassay efficiency of target genes is identical to that of spikedsynthetic RNA, even when sequences are different. This also indicatesthat the definite quantity of target mRNA can be determined by dividingthe values obtained by TaqMan assay with percent recovery of single doseof spiked control RNA in each sample.

Intra-assay variation was approximately 10-20%, as shown in FIGS. 13 and14. In order to assess inter-assay variation, seven differentexperiments were conducted by using the same frozen blood aliquots, inaddition to fresh or frozen blood from the same individual. In eachexperiment, different filterplates and GenePlates were used, and freshmaterials were prepared for lysis, cDNA synthesis, and PCR. As shown inTable III below, percent recovery of control RNA was 4-29%. When wecompared the amounts of CD4 without considering control RNA recovery,these values varied widely. However, after adjusted with percentrecovery in each sample, the values became very similar with aninter-assay CV of 7-14%.

TABLE II Summary of spiked RNA recovery and CD4 mRNA quantitation SpikedRNA CD4 standard curve standard curve copies/μL Subject Fresh/Frozen A*B* % Recovery A* B* copies/μL (adjusted) #1 Frozen −0.18 10.06 12.29 ±0.34 −0.24 13.00   805,901 ± 163,607 6,534,803 ± 1,168,123 #1 Frozen−0.19 10.21  4.70 ± 0.37 −0.23 12.95   358,894 ± 56,669 7,613,541 ±657,139 #1 Frozen −0.20 10.47 11.79 ± 1.31 −0.20 12.40   822,329 ±422,666 7,240,998 ± 4,486,279 #2 Fresh −0.17  9.82 29.13 ± 1.17 −0.1711.15 1,731,727 ± 90,901 5,943,401 ± 170,939 #2 Fresh** −0.20 10.47 4.26 ± 0.88 −0.20 12.40   357,278 ± 252,136 5,524,693 ± 3,434,592 #2Frozen −0.20 10.47 12.27 ± 1.62 −0.20 12.40   557,028 ± 220,0874,477,879 ± 1,384,554 *Copy number = 10{circumflex over ( )}(AxCt + B)**Second blood was drawn from the same individual 2 days after the firstone.

FIGS. 13A-13D demonstrate assay validation. TaqMan assays were conductedfrom cDNA derived from 50 μL of heparinized human whole blood (FIGS.13A, B, D) or synthetic control RNA (FIG. 13C). FIG. 13A showshybridization kinetics. Blood aliquots were frozen at −80° C. Identicalblood aliquots were thawed at different times, and applied tofilterplates to adjust the hybridization length from 30 to 270 minutesat room temperature. FIG. 13B shows dose responses. Blood was dilutedwith PBS 10, 100, and 1000 folds, and 50-200 μL samples were applied tofilterplates. FIG. 13C shows hybridization temperature. Hybridizationwas conducted at 4, 15, 25, and 37° C. for 2 hours. FIG. 13D shows thestability of heparinized whole blood. After blood samples were stored at4 or 37° C. for various lengths of time, each sample was individuallyfrozen. Samples were thawed simultaneously and applied to filterplates.The data were expressed as the mean±standard deviation.

FIGS. 14A-14D represent recovery of synthetic spiked RNA. In FIGS. 14Aand 14B, 0-10¹⁰ copies of RNA34 were applied to each well. Afterconducting mRNA purification and cDNA synthesis, the amounts of controlRNA were determined by TaqMan PCR. FIG. 14A shows the amounts (copynumber) of recovered control RNA versus the amounts of added controlRNA. FIG. 14B shows percent recovery, which was calculated as follows:% Recovery=Amounts of recovered control RNA/amounts of added controlRNA×100.

FIGS. 14C and 14D show percent recovery in the presence of Oligo-dA.After 50 μL of heparinized blood was applied to Leukosorb filterplates,lysis buffer containing various amounts of control RNA34 (□), controlRNA36 (◯), and control bcr-abl (Δ) were applied to each well along with0-10¹⁵ copies of oligo-dA₂₀. After mRNA purification and cDNA synthesis,the amounts of control RNA were determined by TaqMan PCR. FIG. 14C showsthe amounts (copy number) of recovered RNAs versus the amounts ofoligo-dA₂₀. In FIG. 14D the percent total was calculated as follows:% Total=Amounts of recovered RNA with oligo-dA₂₀/amounts of recoveredRNAs without oligo-dA₂₀×100.

Example 6

Fifty μL of heparinized human blood were applied to a filterplate, wherefour layers of Leukosorb membranes were attached. The blood was vacuumaspirated and washed with 150 μL of 5 mM Tris (pH 7.4) twelve times. Thefilterplate was then placed on a GenePlate, and 40 μL of lysis buffer(with or without antisense primer for CD4) were applied to each well.Cell lysates were transferred from the filterplate to the GenePlate bycentrifugation. This process was repeated once with 40 μL of lysisbuffer. After the GenePlate was incubated at room temperature for 2hours, each well was washed with 100 μL of Lysis Buffer three times,followed by three applications of 150 μL wash buffer. The cDNA wassynthesized in each well of the GenePlate by adding cDNA synthesisbuffer and appropriate enzymes. After 37° C. incubation for two hours,each well was washed three times with 150 μL of 95° C. water. Then, CD4mRNA was detected in the GenePlate by TaqMan real time PCR.

In order to validate the hypothesis that oligo(dT) displacesspecifically-primed (by NNNN) cDNA, cDNA was synthesized in theGenePlate with or without specific primers. Then, the CD4 gene wasamplified directly from the GenePlate. As shown in FIG. 17, CD4 wasamplified from both samples. This suggests that upstream cDNA isdisplaced from immobilized oligo(dT)-derived cDNA.

Example 7

Overall Scheme. As illustrated in FIG. 18A, whole blood is applied tofilterplates to trap leukocytes (I). After washing the filterplates withphosphate buffered saline (PBS, Invitrogen), erythrocytes and plasmacomponents are removed. This process is simpler and higher throughputthan that of conventional density gradient separation of peripheralblood mononuclear cells (PBMC). As shown in FIG. 18B, (standard RNA (◯),CD4 (●), p21 (▴), FasL (♦), and leukotrien C4 synthese mRNA (LTC4S) (▪))the filterplate's hybridization performance was slightly better thanthat of the density gradient method. The levels of leukotrien C4synthese (LTC4S) mRNA was significantly less in PBMC, possibly due tothe elimination of the granulocyte population. However, p21 mRNA wassignificantly higher in PBMC, due to secondary induction during thelengthy separation processes (FIG. 22). Another method was compared,wherein whole blood was centrifuged and the pellets were suspended inhypotonic solution (10 mM KHCO₃, 15 mM NH₄Cl, 0.14 mM EDTA, pH 7.2) toburst the erythrocytes, followed by immediate centrifugation toprecipitate the leukocytes (FIG. 18B: Hypotonic). However, the levels ofCD4, p21, FasL, and LTC4S were all less than those of the filterplatemethod.

As shown in FIG. 18A (II), the next step was to apply lysis buffer tothe filterplates and to transfer the lysate to oligo(dT)-immobilizedmicroplates for mRNA purification. In order to evaluate this process,three other methods were employed for comparison. Phenol/guanidineisocyanate (Trizol, Invitrogen) (FIG. 18C: P/GI) or kit-supplied LysisBuffer (RNeasy, Qiagen) (FIG. 18C: Silica) was applied to each well ofthe filterplates, followed by the precipitation of RNA (P/GI) or elutionof RNA from a spin column (Silica) according to the instruction manualsof the products. For direct purification of poly(A)+ RNA, lysis bufferwas applied to the filterplates, and lysates were transferred to eitheroligo(dT)-immobilized microplates (GenePlate, RNAture) (FIG. 18C: dT MP)or fresh microtubes, which contain oligo(dT) cellulose (Invitrogen)(FIG. 18C: dT C). While P/GI, Silica, and dT C methods exhibitedsufficient performance when large amounts of isolated PBMC was used inmicrotubes, these methods did not work well with the filterplate system,where only 50 μl blood was used (FIG. 18C). Moreover, when cell pelletsare lysed in tubes, the degree of mechanical strength (vortex orpipetting) is critical to release mRNA, and this process createssubstantial variation. However, with the filterplate method cells weredispersed within the membrane, and application of lysis buffer wassufficient enough to work without the need for any added mechanicalforce.

Since the lysis buffer preferably contains a mixture of primers, twoindependent hybridization reactions took place simultaneously (FIG.18A). One occurred between immobilized the immobilized oligo(dT) and thepoly(A) tails of mRNA. The other hybridization reaction took placebetween specific primers and appropriate sites in mRNA (FIG. 18A (II)).Although the design of specific primers is critical, sufficienthybridization time made the assay more reproducible than that of primerhybridization during cDNA synthesis. It first appeared as thoughcDNA-mRNA duplex stayed in the solid surface via hybridization withimmobilized oligo(dT) (FIG. 18A (II)). Thus, cDNA was removed from thesolid surface by heating at 95° C. for 5 min. However, the amounts ofamplified genes were unchanged to those of un-heated control (FIG. 18Dinset). To test whether the cDNA-mRNA duplex was somehow removed fromthe solid surface, microplates were washed with water extensively aftercDNA synthesis, and used for PCR directly. However, the target gene wassuccessfully amplified from microplates with or without specific primersduring the hybridization step (FIG. 18D). These data suggest that thespecific primer-primed cDNA may be displaced with oligo(dT)-primed cDNA(FIG. 18A (III, IV)). This makes the system advantageous; because thecDNA in solution is used for gene quantitation, the microplate itselfcan be used as a cDNA bank for validation, storage, and future use.

In order to validate RNA stability during lysis and subsequenthybridization processes, various concentrations of lysis buffercontaining equal amounts of standard RNA were diluted with water orconcentrated eosinophil extract. As shown in FIG. 18E, the eosinophilextract itself largely abolished standard RNA quantitation when it wassuspended with wash buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 0.5 M NaCl),where hybridization stringency was maintained without major lysis buffercomponents. However, when eosinophil extract was suspended in the lysisbuffer, RNA was maintained, and was similar to that of water dilution(FIG. 18E). The wide range of optimal lysis buffer concentrations(70-120%) made this system robust and reproducible. Lysis bufferconcentrations higher than 140% significantly reduced the assayperformance.

Assay optimization. Studies were conducted to identify the optimal,reproducible conditions that exhibit equal performance among fivedifferent target RNAs, as illustrated in FIGS. 19A-F (standard RNA (◯),CD4 (●), p21 (▴), FasL (♦), and leukotrien C4 synthese mRNA (LTC4S)(▪)). The reproducible conditions are critical for the subsequent genequantitation section. Each data point in FIG. 19 is the mean±standarddeviation (s.d.) from triplicate blood aliquots (50 μl each) from asingle typical experiment. However each experiment was reproduced atleast two to three times. First, three typical anticoagulants weretested. Although heparin exhibited slightly better performance than ACDand EDTA, all 3 anticoagulants are acceptable (FIG. 19A). Since ACD andEDTA chelate calcium, which is a critical component for many biologicalactivities, heparin was the choice of anticoagulant of this projectwhere whole blood will be used for stimulation in vitro (FIG. 22).

Maintaining the stability of whole blood after drawing blood is a primeconcern. Thus, some commercial systems (PAX gene, PreAnalytix) usesspecial blood container, where cells are lysed immediately, and releasedRNA is stabilized for a relatively long period. However, manipulation oflarge volumes of lysate make entire systems problematic. Moreover,because one of the goals of this project is to quantitate mRNA beforeand after gene induction processes in vitro (FIGS. 22A-H), heparinizedwhole blood was stored at 4° C. and the changes in mRNA levels wereexamined. Although the levels of four native genes (CD4, p21, FasL, andLTC4S) were not stable after the blood draw, the levels became stableand constant after two hours whenever blood was stored at 4° C. (FIG.19B).

Poly(A)+ mRNA preparation with oligo(dT) solid surface is usuallyconducted at room temperature. However, the performance varies between20 and 30° C. (FIG. 19C). When short synthetic RNA was used, thedifference between 20 and 23° C. was significant (FIG. 19C). Thus, mRNApreparation step was conducted at 4° C. The length of hybridization wasalso critical. Some RNAs (standard RNA and FasL) reached a plateau aftertwo hours, whereas others required more than four to eight to stabilize(FIG. 19D). Consequently, the mRNA preparation step was conducted at 4°C. overnight. By switching to this condition, assay-to-assay variationwas substantially improved.

The cDNA was synthesized without any additional primers (FIG. 18D).Although short synthetic RNA and abundant RNA (CD4) required smallamounts of reverse transcriptase, other pieces of RNA requiredapproximately 100 units of MMLV reverse transcriptase to reach to aplateau (FIG. 19E). Interestingly RnaseH⁻MMLV (Superscript, Invitrogen)exhibited poor performance compared to that of native MMLV in thissystem. More than 90 min incubation at 37° C. was enough for all speciesof RNA tested (data not shown).

The cDNA in solution was used directly for subsequent TaqMan real timePCR. The assay becomes sensitive in proportion to the amounts of cDNAtransferred to PCR. Commonly available buffers contain dithiothreitol(DTT), which inhibits PCR. Thus, as shown in FIG. 19F, maximal cDNAvolume was 2 μl per 10 μl PCR. By removing DTT from buffer, the volumeof cDNA increased to 4 μl per 10 μl PCR.

Quantitation. The goal in the quantitation step of this study was todetermine total assay efficiency in each sample by using known amountsof spiked standard RNA, which is further used as a denominator toconvert PCR results to the quantity of target mRNAs in original samples.The principle relies on two assumptions: that the efficiency isidentical between samples of similar mRNA with varying abundancies(dose-independence); and that the efficiency is identical amongdifferent mRNA sequences (sequence-independence). In order to validatethe first assumption, different amounts of synthetic RNA-standard wasadded to the lysis buffer, which was exposed to filterplates containing50 μl of blood. After it was confirmed that standard RNA was notamplified from human blood alone, the recovery of standard RNA wasdetermined by TaqMan PCR. As shown in FIG. 20A (standard RNA (◯), CD4(●), p21 (▴), FasL (♦), and leukotrien C4 synthese mRNA (LTC4S) (▪)),dose-dependent recovery of standard RNA was observed at the tested rangeof 10⁴ to 10⁹ molecules well. Since this range of standard RNA was smallenough compared to the amounts of total mRNA existing in 50 μl of blood,the levels of the four other native mRNAs maintained unchanged (FIG.20A). When the same data were converted to percent recovery, thesevalues all became similar around 2-3% (FIG. 20B). Under the equilibriumconditions of hybridization, the dissociation constant (Kd) wascalculated as followed:Kd=[RNA]×[oligo(dT)]/[RNA:oligo(dT)]

where [RNA] and [oligo(dT)] represent the concentrations of unboundstates of RNA and oligo(dT), respectively, and [RNA:oligo(dT)]represents the concentrations of hybridized RNA with oligo(dT). Thismeans that the Kd is maintained as a constant value in this system, and[RNA:oligo(dT)] changes depending on the amounts of applied RNA (FIG.20A). In fact, [RNA:oligo(dT)] was increased in proportion to theamounts of applied RNA, when hybridized whole RNA was directly measuredby Yoyo-1 nucleic acid dye. These data also suggest that the percentrecovery derived from one concentration of standard RNA can beapplicable to any concentration of mRNA within the same samples.

For the second assumption, hybridization was carried out with or withoutoligo(dA) as a competitive inhibitor. As shown in FIG. 20C, the Ctvalues of all five target RNAs were significantly inhibited by oligo(dA)at more than 3×10¹² molecules per well, although the expression levelsof these RNAs were all different. Interestingly, when the same data weretransformed to the percent inhibition, all five RNAs showed almostidentical inhibition curves with an IC₅₀ around 3×10¹²-10¹³ moleculesper well (FIG. 20D). This indicates that the system is poly(A)-specific,and sequence-independent. As shown in FIG. 20C, some non-specificactivities remained even after 10¹⁵ molecules of oligo(dA) were applied(standard RNA and CD4). However, as shown in FIG. 20D, thesenon-specific activities were negligible, when the Ct values (log scale)were converted to the number of molecules (linear scale). Thus,non-poly(A) sequences do not appear to play major role in this system.Because FIG. 20A-D were the sum of whole processes of mRNA quantitation,these data suggest that the total assay efficiency of any target gene isidentical to that of spiked standard RNA. This is unique to poly(A)⁺RNA, because substantial variation exists between long and short RNA inconventional total RNA purification methods (data not shown).

The next step was to convert the amounts of RNA in each well to theamounts of RNA per μl of blood. As shown in FIG. 20E, the Ct values offour native mRNAs were decreased almost linearly, depending on thevolume of blood applied. Abundant mRNA such as CD4 was detectable fromeven 0.001 μl of blood (1:10⁵ dilution, 100 μl per well) (FIG. 20E).Since the amount of standard RNA in lysis buffer was identical, therecovery of standard RNA was unchanged even when the blood volume variedwidely FIG. 20E. The Ct values reached a plateau at more than 100 μlblood per well (FIG. 20E), suggesting increased leakage of leukocytesfrom filterplates. Once the same data were transformed to amounts ofμl⁻¹ blood, the values were consistent between 3 and 50 μl blood perwell (FIG. 20F), which was true for all four native mRNAs (FIG. 20F).

The quantitation also relies on two independent absolute values: thequantity of applied standard RNA, and the quantity of standard DNAtemplates in TaqMan PCR. To ensure the purity of the standard RNAproducts, RNA oligonucleotides were used in this project. While thesynthesis of RNA oligonucleotides with a length of 100 bases long can bedifficult to achieve, such a length is desirable because RNAoligonucleotides preferably comprises two primer sites, a TaqMan probesite, and a poly(A) tail. Synthetic RNA in the present study wassynthesized by Dharmacon with a purity of 86% by HPLC analysis.HPLC-purified DNA oligonucleotides were also used as templates forTaqMan PCR because the slope of the amplification curve (showing PCRefficiency) was identical between oligonucleotides and cDNA. A standardcurve was generated with 10⁶-10 molecules of oligonucleotides per well.A problem occurred during 10⁶ to 10¹² times dilution of μMconcentrations of stock solution. When TE or water was used as adiluent, the standard curve varied widely; particularly with less than10³ molecules per well. After switching to nuclease-free watercontaining 0.1% tween-20, this problem was completely eliminated.

Determining Normal Values. In order to determine the control values ofmRNA per μl of blood in healthy subjects, the levels of CD4, p21, FasL,and LTC4S were measured from 52 individuals (54 data points, with oneindividual repeated three times) over two months, through 15 differentexperiments. Each data point was derived from three aliquots of 50 μlwhole blood. As shown in FIG. 21A, the recovery of standard RNA was3.56±0.49% (CV=13.7%). Although this CV value is much larger than aconventional immunoassay, it is acceptable because it uses PCR, whereone cycle difference represents a doubling in product quantity. Usingthe values of standard RNA recovery, the data of each mRNA weresuccessfully converted to the number of molecules per μl of blood,rather than relying on pico moles or femto moles.

As shown in FIGS. 21B-E (standard RNA (◯), CD4 (●), p21 (♦), FasL (♦),and leukotrien C4 synthese mRNA (LTC4S) (▪)), control levels of CD4,p21, FasL, and LTC4S mRNA were 100,772±59,184 (CV=58.7%), 1,692±858(CV=50.7%), 17,841±12,190 (CV=68.3%), and 42,058±22,521 (CV=53.5%)molecules per μl of blood, respectively. Fifty percent CV means that thenormal values reside within one Ct in TaqMan PCR. Interestingly, whenthe mean±1 s.d. values were blanketed in each figure, some individualsexpressed high values (FIGS. 21B-E). One individual who expressed highFasL mRNA levels was reproduced three times (FIG. 21D). Determination ofnormal values of mRNA, combined with low CV values and standardizationobtainable by preferred embodiments of the present invention, can beused in assays for the detection of various diseases known to thoseskilled in the art.

In vitro Responsiveness. In order to assess leukocyte responsivenessagainst phorbol 12-myristate 13-acetate (PMA) and calcium ionophoreA23187 (CaI) (Sigma) as a model system, the levels of p21 and FasL mRNAwas quantitated (FIG. 22). In other preferred embodiments, various typesof mRNA can be analyzed in response to stimulation by various bioactiveagents, including but not limited to, for example: radiation,ultraviolet, oxidative stress, ozone, temperature, mechanical stress,chemicals, peptides, hormones, proteins, antigens, antibodies, drugs,small molecule compounds, toxic materials, environmental stimuli,cell-cell communications, infectious agents, and allergens. Since thesystem used heparinized whole blood, rather than an isolated leukocytesuspension in artificial solution, the results reflected physiologicallyaccurate conditions. In FIGS. 22A-H, Δ shows p21 mRNA for the controlstimulation, ▴ shows p21 mRNA for the PMA+CaI stimulation, ⋄ shows FasLmRNA for the control stimulation, and ♦ shows FasL mRNA for the PMA+CaIstimulation. As shown in FIG. 22A, both p21 and FasL mRNA levelsincreased rapidly upon stimulation of PMA and CaI, and reached a plateauafter 90-120 minutes with approximately a ten-fold increase. Theincreases in p21 were much faster than those of FasL (FIG. 22A). Thelevels of p21 were also increased slightly by incubation at 37° C.without any stimulation, whereas FasL remained unchanged (FIG. 22A).Interestingly, the responsiveness was preserved even when heparinizedwhole blood was stored at 4° C. for 21 hours (FIG. 22B), which provideswide flexibility for functional molecular analysis.

In analyzing up- or down-regulation of mRNA expression, fold-increase isa commonly used parameter. However, as shown in FIGS. 22C-D, the amountsof p21 and FasL mRNA induced after PMA-CaI stimulation increasedlinearly depending on the amounts of basal levels of mRNA. Thus, thefold-increase measurement was not capable of identifying non-ordinarysamples that resided on the regression line (FIG. 22G), even when thesample was more than two standard deviations apart from the normalpopulation (FIG. 22C). The fold-increase measurement identified anabnormal sample (FIG. 22H) that resided away from the regression line(FIG. 22D). The two dimensional graphs of FIGS. 22C-D clearlydistinguished ordinary and non-ordinary samples in both cases. In FIGS.22C-H, each data point is the mean from triplicate determinations. Inorder to make the graph simple, the standard deviation was not shown.However, because the blanket areas with mean±2 s.d. in both X- andY-axes (FIGS. 22C-D) contained open spaces in the upper left and lowerright corners, non-ordinary samples in these corners were difficult toidentify. By rotating the X-axis to the regression line (FIGS. 22E-F),the size of the blanketed areas were minimized. These graphs (FIGS.22E-F) provide a better way to detect non-ordinary samples out of thenormal population.

1. A method of high throughput quantification of a specific mRNA inwhole blood, comprising the following steps in order: (a) obtaining ablood sample by a method consisting of: (i) collecting whole blood in acontainer consisting of a blood collection tube, wherein, prior to thecollecting step, the content of the blood collection tube consists of ananticoagulant; (ii) freezing the whole blood; and (iii) thawing thewhole blood; (b) lysing leukocytes of the whole blood thereby producinga lysate comprising mRNA; wherein said lysing step is performed with alysis buffer which inhibits RNases; and (c) quantifying the specificmRNA in the lysate.
 2. The method of claim 1, wherein erythrocytes andblood components other than leukocytes are removed from the whole bloodbefore the lysing step.
 3. The method of claim 2, wherein theerythrocytes and blood components other than the leukocytes are removedby filtration and the leukocytes are captured on a filter membrane; andthe leukocytes are lysed on the filter membrane.
 4. The method of claim1, wherein, after the lysing step, the lysate is transferred to anoligo(dT)-immobilized plate to capture the mRNA.
 5. A method of highthroughput quantification of a specific mRNA in whole blood, comprisingthe following steps in order: (a) obtaining a blood sample by a methodconsisting of: (i) collecting whole blood in a container consisting of ablood collection tube, wherein, prior to the collecting step, thecontent of the blood collection tube consists of an anticoagulant; (ii)stimulating the whole blood with a bioactive agent; (iii) freezing thewhole blood; and (iv) thawing the whole blood; (b) lysing leukocytes ofthe whole blood thereby producing a lysate comprising mRNA; wherein saidlysing is performed with a lysis buffer which inhibits RNases; and (c)quantifying the specific mRNA in the lysate.